Eukaryotic expression vectors are utilized for various biomedical applications including protein production (in eukaryotic cell lines) from transient or integrated cell lines, and in vivo gene therapy, transgenic or vaccination applications.
A key barrier in this technology is that expression vectors maintained transiently, or stably integrated, undergo promoter inactivation (silencing) over time.
Transgene silencing of non-integrating vectors such as plasmid, AAV, adenoviral vectors, etc may be mediated by the prokaryotic region of the vector which has been linked to transgene silencing of plasmid vectors in transfected cell lines and in tissues in vivo (Chen Z Y, He C Y, Meuse L, Kay M A. 2004. Gene Therapy 11: 856-864).
Gene silencing is also a critical problem with stably integrated nonviral vectors (e.g. Sleeping beauty transposon; Garrison B S, Yant S R, Mikkelsen J G, Kay M A. 2007. Molecular Cellular Biology 27: 8824-8833) and viral vectors such as retroviral (Katz R A, Jack-Scott E, Narezkina A, Palagin I, Boimel P, Kulkosky J, Nicolas E, Greger J G, Skalka A M. 2007. J. Virol. 81: 2592-2604) and lentiviral (Nielsen T T, Jakobsson J, Rosenqvist N, Lundberg C. 2009. BMC Biotech. 9:13) vectors that also undergo promoter inactivation (silencing) over time. Silencing with these vectors is mediated by genomic DNA flanking the insertion site since the prokaryotic region of the vector is not integrated into the genome.
This lack of sustained transgene expression negatively impacts the cost of cell culture production of recombinant proteins, and has also limited the in vivo application of non-viral and viral vector systems to short term applications.
Various technologies have been developed to attempt to address this limitation.
Insulator elements: One strategy to reduce transgene silencing is to include boundary or insulator elements (e.g. scaffold or matrix attachment regions; reviewed in West A G, Gaszner M, Felsenfeld G. 2002. Genes Dev 16: 271-288) in the vector to insulate the transgene from integration site dependent inactivation. Multiple vector systems incorporating matrix attachment regions (MARs) have been developed and demonstrated to have utility. For example, incorporation of the Chicken Lysozyme 5′ matrix attachment region into a plasmid has been shown to improve transgene expression in vitro with integrated and transient Chinese hamster ovary (CHO) cell culture systems (Girod P A, Zahn-Zabal M, Mermod N. 2005. Biotechnol. Bioeng. 91: 1-10) and in vivo from plasmid based non-viral vectors (Ehrhardt A, Peng P D, Xu H, Meuse L, Kay M A. 2003 Hum Gen. Ther 14: 215-225).
Minicircle vectors: An alternative strategy to prevent transgene inactivation is to remove the prokaryotic region of the plasmid. For example, minicircle and linear ‘Minimalistic immunogenic defined gene expression’ (Midge) vectors have been developed which do not contain a prokaryotic region. Removal of the prokaryotic region in minicircles improved transgene expression in transfected cell lines (Suzuki M, Kasai K, Saeki Y. 2006. J. Virol. 80: 3293-3300) and in animals (Chen et al., Supra. 2004).
Transgene silencing mechanisms: Non replicating transiently transfected plasmids are not inactivated by de novo methylation; this is not surprising, since methylation typically requires replication. Rather, the bacterial sequences within the vector appear to become associated with an inactive form of chromatin which then triggers transcriptional silencing of the entire vector (Suzuki et al., Supra, 2006). One strategy to reduce this effect is to clone a locus control region into the vector as described above. This partially alleviates silencing by blocking transcriptional inactivation (Miao C H, Thompson A R, Loeb K, Ye X. 2001. Mol. Ther. 3: 947-957). This data would indicate that the improvement observed with MARs and minicircle vectors are due to a similar mechanism: prevention of prokaryotic region mediated silencing by blockage (MARs) or removal (minicircles).
Current barriers: Methods to manufacture midge and minicircle vectors are expensive and not easily scalable. For example, optimal manufacture of minicircle DNA vectors yields only 2 mg of minicircle per liter culture (Chen Z Y, He C Y, Kay M A. 2005. Hum Gene Therapy 16: 126-131) compared to 2200 mg/L with optimized plasmid vectors and an inducible fermentation process (Williams, J. A., Luke, J., Langtry S., Anderson, S., Hodgson, C. P., and Carnes, A. E. (2009). Biotechnol Bioeng 103:1129-1143).
Matrix attachment regions are large (the commonly utilized chicken lysozyme 5′ MAR is 3 kb; Girod et al., Supra, 2005); inclusion of this region therefore dramatically reduces the potency of a plasmid. As well, MARs are not a general solution since they improve expression only in certain cell lines (Chancham P, van Ljperen T, McDoom I, Hughes J A. 2003. J Drug Targeting 11: 205-213). Incorporation of large MAR sequences into limited payload integration vectors such as retroviral, lentiviral and transposon vectors is not feasible, and some complex chromatin insulators such as the chicken β globin insulator block lentiviral transduction (Nielsen et al., Supra, 2009).
There is clearly a significant need for more efficient methods to prevent prokaryotic region mediated transgene silencing of eukaryotic expression plasmids.